Rapid development of ultrasound contrast agents in recent years has generated a number of different formulations, which are useful in ultrasound contrast imaging of organs and tissue of a human or animal body. These agents are designed to be used primarily as intravenous or intra-arterial injectables in conjunction with the use of medical echographic equipment which employs for example, B-mode image formation (based on the spatial distribution of backscatter tissue properties) or Doppler signal processing (based on Continuous Wave or pulsed Doppler processing of ultrasonic echoes to determine blood or liquid flow parameters).
A class of injectable formulations useful as ultrasound contrast agents includes suspensions of gas bubbles having a diameter of a few microns dispersed in an aqueous medium.
Of particular interest are gas bubbles which are stabilized by means of suitable additives such as, for example emulsifiers, oils, thickeners or sugars, or by entrapping or encapsulating the gas or a precursor thereof in a variety of systems. These stabilized gas bubbles are generally referred to in the art as “microvesicles”, and may be divided into two main categories.
A first category of stabilized bubbles or microvesicles is generally referred to in the art as “microbubbles” and includes aqueous suspensions in which the bubbles of gas are bounded at the gas/liquid interface by a very thin envelope (film) involving a stabilizing amphiphilic material disposed at the gas to liquid interface. Microbubble suspensions are typically prepared by contacting powdered amphiphilic materials, e.g. freeze-dried preformed liposomes or freeze-dried or spray-dried phospholipid solutions, with air or other gas and then with an aqueous carrier, while agitating to generate a microbubble suspension which can then be administered, preferably shortly after its preparation.
Examples of aqueous suspension of gas microbubbles and preparation thereof are disclosed, for instance, in U.S. Pat. Nos. 5,271,928, 5,445,813, 5,413,774, 5,556,610, 5,597,549, 5,827,504, WO 97/29783 and in co-pending International Patent Application PCT/IB04/00243, which are here incorporated by reference in their entirety.
A second category of microvesicles is generally referred to in the art as “microballoons” or “microcapsules” and Includes suspensions in which the bubbles of gas are surrounded by a solid material envelope of a lipid or of natural or synthetic polymers. Examples of microcapsules and of the preparation thereof are disclosed, for instance, in U.S. Pat. No. 5,711,933 and U.S. Pat. No. 6,333,021, herein incorporated by reference in their entirety.
Microvesicles preparations are characterized, among other factors, also by their respective mean size and size distribution (which gives an Indication on how the microvesicle population is scattered around the mean size). Size-distributions of microvesicles preparations can in general be assimilated to a Gaussian-like distribution, centred on the mean size value thereof.
Contrast imaging is based on the ability of gas-filled microvesicles to resonate when hit by an ultrasound wave emitted by an ultrasound probe at a certain frequency, thus reflecting a corresponding echo signal which is detected by the ultrasound probe and then imaged. As the echo response of a contrast agent is rather peculiar with respect to the echo response of tissues or organs itself, the contrast agent contained in the vessels can be easily imaged with respect to the surrounding tissue or organ. The resonance capacity of a gas-filled microvesicle depends, among other factors, also from the compatibility of its size with the frequency of the transmitted radiation. As a general indication, smaller microvesicles resonate at higher frequencies, while larger microvesicles resonate at lower frequencies. In addition, the intensity of a reflected echo is in general proportional to the concentration of microvesicles having said predetermined compatible dimensions, said concentration being for instance expressed as the total volume of gas entrapped in said microvesicles.
The Applicant has now observed that, for a specific contrast agent, it is possible to define a preferred size range and a corresponding size distribution which is suitably responsive to a determined transmission frequency. As observed by the Applicant, at low frequencies (e.g. from about 1.5 to about 3.5 MHz), said size distribution typically has a relatively large median diameter (e,g. DV50 of about 4 μm) and is in general relatively broad; this observation is consistent with the fact that conventional broadly distributed gas-filled microvesicles can in general be employed for the contrast imaging at these low frequencies, as a sufficiently large number of microvesicles are available for resonating when hit by the selected low frequency ultrasound wave. On the other side, at higher frequencies (e.g. 5 MHz or higher), the size distribution of suitably responsive microvesicles substantially narrows. In addition, said narrow distribution is generally associated with a corresponding relatively smaller DV50 value (e.g. from about 1 to 2.5 μm), in accordance with the fact that small microvesicles resonate at higher frequency. This observation is also consistent with the fact that conventional broadly distributed microvesicle preparations are in general much less responsive at high frequency contrast imaging, as the fraction of small dimensions microvesicle contained therein is relatively low. Thus, when using high transmission frequencies for ultrasound imaging, suitably calibrated gas-filled microvesicle preparations having relatively narrow size distributions with relatively small median dimensions (DV50, in particular) shall be employed for an effective contrast imaging, said preparations being however not as effective when used at low transmission frequencies.
In general, relatively low transmission frequencies (e.g. 0.5-2 MHz) are employed for echographic analysis in deep body regions, such as for cardiac applications, while relatively high transmission frequencies (e.g. 5-7 and up to 10-15 MHz) are generally employed for abdominal (e.g. kidney, liver etc.) or superficial analysis (e.g. ophthalmology, breast analysis etc.). Higher transmission frequencies (e.g. 15-20 MHz and up to 80 MHz) can also be employed for specific applications, for instance in intravascular ultrasound imaging.
The Applicant has now found a new composition suitable for providing an effective echo response to at least two selected ultrasound waves having different frequencies. As observed by the Applicant, said effective echo response can be obtained by suitably tailoring the size distribution of a gas-filled microvesicles preparation. Advantageously, said preparation comprises an effective amount of microvesicles having a relatively small size, being thus responsive to a respective relatively high selected transmission frequency, and an effective amount of microvesicles with a relatively larger size, responsive to a respective relatively lower selected transmission frequency, said effective amount of large size microvesicles being nevertheless sufficiently low so as to not excessively attenuate the response of the small size microvesicles at the selected high transmission frequency.
International Patent application WO 98/32468 discloses compositions comprising two or more types of gas containing microparticles having different susceptibility to ultrasonic pressure. Preferred composition are those comprising a first type of microparticles with a relatively soft encapsulating shell (such as microbubbles with phospholipid shells) and a second type of microparticles with relatively hard encapsulating material (such as polymer or protein shelled microcapsules). In particular, example 1 of said patent discloses mixtures of microbubbles comprising hydrogenated egg phosphatidylserine with microcapsules containing a polymer comprising repeating units of formula:—O—(CH2)a—CO—O—CH(CH3)—O—CO—(CH2)a—CO—O(CH2)b—CO—
where a is an integer from 9 to 19 and b is an integer from 1 to 8.
WO01/68150 discloses microcapsules having a stabilizing envelope comprising a polyalkylcyanoacrylate polymer.